Loss of cell cycle checkpoint control and acquisition of the ability to proliferate indefinitely constitute two of the fundamental changes required for the development of cancer. Rak, a non-receptor tyrosine kinase identified through our enhanced retroviral mutation screen as a represser of hTERT function, has been implicated in cellular immortalization. Our preliminary data indicate that Rak has additional functions in the transmission of DNA damage signals and is required for intact DNA damage checkpoints. Moreover, Rak is located on chromosome 6q21-23, a region that undergoes loss of heterozygosity in 30% of breast cancer cases. Rak expression is also significantly reduced in breast cancer tissues and cancer cell lines as analyzed by DNA arrays, real-time PCR, and protein analysis. Thus, we hypothesize that Rak may function as a tumor suppressor gene in breast cancer through dual functions: repressing cellular immortalization and maintaining genomic stability. This hypothesis will be tested in the following steps. (1) We will determine the role of Rak in preventing cellular immortalization by using normal human mammary epithelial cells that conditionally express small hairpin RNA against Rak, and we will investigate the mechanisms mediating Rak's function on hTERT expression. (2) We will use Rak-depleted cells to study the role of Rak in the response to genotoxic stress, maintenance of genomic integrity, and cellular transformation; for these studies, we will assess genomic instability by using comparative genomic hybridization (CGH) and spectral karyotyping (SKY) to visualize chromosomal aberrations; we will study how Rak may participate directly in transmitting DNA damage signals; and we will use a knockout mouse model to assess Rak's function in vivo in maintaining genomic integrity and suppressing tumor formation. (3) We will identify and characterize aberrations in Rak in samples from patients with breast cancer to determine if Rak serves as a prognostic marker or a therapeutic target in cancer. As a corollary, analyzing the function of Rak and its aberrations in patients with cancer will contribute to an improved understanding of the key pathologic alterations in cancer initiation and progression and to the development of novel, effective therapeutic approaches for cancer. Relevance: Rak plays a dual role in preventing cell immortalization and maintaining genomic stability. Our studies have identified Rak as a potentially important tumor suppressor gene in breast cancer. An analysis of the function of Rak and its network of interacting proteins will clarify the key pathologic alterations in cancer initiation and progression and eventually will aid in the development of novel, effective therapeutic approaches for breast cancer.